Fad4, Fad5, Fad5-2 and Fad6, novel fatty acid desaturase family members and uses thereof

• US 7,087,432 B2
• Filed: 09/28/2001
• Issued: 08/08/2006
• Est. Priority Date: 09/28/2000
• Status: Expired due to Term

• Current Assignee: Bioriginal Food & Science Corp. (Omega Protein Corporation)
• Original Assignee: Bioriginal Food & Science Corp. (Omega Protein Corporation)
• Inventors: Hong, Haiping, Qiu, Xiao
• Primary Examiner(s): McElwain, Elizabeth F.
• Application Number:
  US09/967,477
• Publication Number:
  US 20020156254A1
• Time in Patent Office: 1,775 Days
• Field of Search: 536/23.1, 536/23.2, 435/468, 435/471
• US Class Current: 435/468
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• CPC Class Codes: A23L 33/12 Fatty acids or derivatives thereof
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• CPC Class Codes: A61K 31/202 having three or more double bonds, e.g. linolenic eicosanoids, e.g. leukotrienes A61K31/557
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 25/18 Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• CPC Class Codes: A61P 25/24 Antidepressants
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/02 : Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/12 : Antihypertensives
• CPC Class Codes: C12N 15/8247 : involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/0071 : acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/0083 : Miscellaneous (1.14.99)
• CPC Class Codes: C12P 7/6427 : Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6431 : Linoleic acids [18:2[n-6]]
• CPC Class Codes: C12P 7/6432 : Eicosapentaenoic acids [EPA]
• CPC Class Codes: C12P 7/6434 : Docosahexenoic acids [DHA]
• CPC Class Codes: C12P 7/6472 : containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12Y 114/19003 : Linoleoyl-CoA desaturase (1.14.19.3)
• CPC Class Codes: Y02P 20/52 : using catalysts, e.g. selective catalysts

Abstract

The invention provides isolated nucleic acid molecules which encode novel fatty acid desaturase family members. The invention also provides recombinant expression vectors containing desaturase nucleic acid molecules, host cells into which the expression vectors have been introduced, and methods for large-scale production of long chain polyunsaturated fatty acids (LCPUFAs), e.g., DHA.

Claims

1. An isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:

1, or a complement thereof.

4. A method of producing an unsaturated fatty acid comprising transfecting or transforming a cell with the isolated nucleic acid molecule of claim 1, 2, or 3 and culturing or cultivating the cell under conditions such that the unsaturated fatty acid is produced.

5. The method of claim 4, wherein the cell is selected from the group consisting of a plant cell, an animal cell, and a microbial cell.

6. The method of claim 5, wherein the plant is selected from an oilseed crop.

7. The method of claim 6, wherein the oilseed crop is selected from the group consisting of flax (Linum sp.), rapeseed (Brassica sp.), soybean (Glycine and Sola sp.), sunflower (Helianthus sp.), corron (Gossypium sp.), corn (Zea mays), olive (Olea sp.), safflower (Carthamus sp.), cocoa (Theobroma cacca), and peanut (Arachis sp.).

8. The method of claim 4, further comprising the step of recovering the unsaturated fatty acid.

9. The method of claim 4, wherein the unsaturated fatty acid is selected from the group consisting of GLA 18:

3 (6,9,12), SDA 18;

4 (6,9,12,15), AA 20;

4 (5,8,11,14), EPA 20;

5 (5,8,11,14,17), DPA 22;

5 (4,7,10,13,16) and DHA 22;

6 (4,7,10,13,16,19).

10. A method of producing a cell capable of generating an unsaturated fatty acid comprising, introducing into said cell the nucleic acid molecule of claim 1, 2 or 3, wherein the nucleic acid molecule encodes a desaturase having an activity of catalyzing the formation of a double bond in a fatty acyl chain.

11. The method of claim 10, wherein the cell is selected from the group consisting of a plant cell, and a microbial cell.

12. The method of claim 11, wherein the plant cell is a cell from an oilseed plant.

13. The method of claim 12, wherein the oilseed plant is flax (Linum sp.), rapeseed (Brassica sp.), soybean (Glycine and Sola sp.), sunflower (Helianthus sp.), corron (Gossypium sp.), corn (Zea mays), olive (Olea sp.), safflower (Carthamus sp.), cocoa (Theobroma cacoa), and peanut (Arachis sp.).

14. The method of claim 10, wherein the unsaturated fatty acid is selected from the group consisting of GLA 18:

3 (6,9,12), SDA 18;

4 (6,9,12,15), AA 20;

4 (5,8,11,14), EPA 20;

5 (5,8,11,14,17), DPA 22;

5 (4,7,10,13,16) and DHA 22;

6 (4,7,10,13,16,19).

2. An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

2, or a complement thereof.

3. An isolated nucleic acid molecule encoding a Δ

4 fatty acid desaturase molecule capable of introducing a double bond at position 4 of the fatty acid molecule 22;

5(7,10,13,16,19) to form a Δ

4 desaturated fatty acid molecule DHA (22;

6(4,7,10,13,16,19)), or a complement thereof, wherein the desaturase molecule comprises an amino acid seciuence which is at least 70% identical to the amino acid sequence set forth in SEQ ID NO;

2 and comprises a heme-binding motif and a histidine-rich motif.

17. The isolated nucleic acid molecule of claim 3, wherein the desaturase molecule comprises at least one motif represented by the amino acid residues selected from the group consisting of residues 41–

44, residues 182–

186, residues 216–

223 and residues 453–

462 of SEQIDNO;

2.

18. The isolated nucleic acid molecule of claim 17, wherein the desaturase molecule comprises at least two conservative histidine motifs.

15. An isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO:

1, or a complement thereof, wherein the nucleic acid molecule encodes a Δ

4 fatty acid desaturase molecule capable of introducing a double bond at position 4 of a fatty acid molecule.

16. An isolated nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence which is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:

2, or a complement thereof, wherein the polypeptide is a Δ

4 fatty acid desaturase molecule capable of introducing a double bond at position 4 of a fatty acid molecule.

19. An isolated nucleic acid molecule encoding a Δ

4 fatty acid desaturase molecule capable of introducing a double bond at position 4 of the fatty acid molecule 22;

5(7,10,13,16,19) to form a Δ

4 desaturated fatty acid molecule DHA (22;

6(4,7,10,13,16,19)), or a complement thereof, wherein the desaturase molecule comprises an amino acid sequence which is at least 70% identical to the amino acid sequence set forth in SEQ ID NO;

2 and further comprises amino acid residues 41–

44, residues 182–

186, residues 216–

223 and residues 453–

462 of SEQ ID NO;

2.